Antisense RNAs are small diffusible transcripts which lack coding capacity and bind to complementary regions of a target RNA causing suppression of gene expression (2). It has been suggested that inhibition of expression occurs by RNA duplex formation (2) which in turn inhibits transport from the nucleus to cytoplasm (3) or prevents translation (4). Antisense RNAs were initially recognized in bacteria as a naturally occurring mechanism for regulation of gene expression (5). This later led to the design of artificial antisense control strategies and recently, this technology has been used for the regulation of gene expression in plants.
Plants were the first multicellular organisms in which antisense transcripts have been experimentally applied to suppress expression of endogenous genes (6). All reported antisense gene regulation in plants known to the inventors, have made use of RNAs transcribed by RNA polymerase II, particularly from the strong cauliflower mosaic virus (CaMV) 35S RNA promoter (6-15). Success has varied dependent upon the target gene and the context in which expression is studied. The inability to express sufficient levels of antisense RNAs frequently has led to incomplete and ineffective gene regulation. Alternate polymerase II promoters have been employed by some investigators seeking to improve the effectiveness of gene suppression.
An alternative approach for expressing high levels of RNA transcripts is the use of RNA polymerase III transcription units. RNA polymerase III is found in all cells where it synthesizes abundant transcripts (16). The tRNA methionine initiator (tRNA.sup.met.sub.i) gene encodes the initiator tRNA utilized for virtually all cytoplasmic protein synthesis in plants and consequently is expressed in every plant tissue (17). In mammalian cells, tRNA.sup.met.sub.i species represents 5% of all tRNAs and it is estimated that the number of transcripts expressed from one of the genes is equal to approximately 25% of the total polyadenylated RNA present in the cell (18). The tRNA.sup.met .sub.i gene is therefore very active making it well suited for antisense expression.
Antisense RNAs were first engineered to be synthesized by RNA polymerase III with co-transcription by the adenovirus VA1 gene (19). Expression of this gene linked to SV40 virus antisense sequences in monkey cells resulted in transient inhibition of SV40-replicon function by greater than 50% and demonstrated that RNA polymerase III can effectively express RNA. More recently, it has been shown that the human tRNA.sup.met.sub.i gene fused to antisense templates, when introduced into animal cells, was capable of inhibiting replication of Moloney murine leukemia virus (MoMLV) by 97% and replication of HIV-1 virus by 99% (20,21).
There is no predictability between the function of antisense messages in animal studies, especially studies in cell lines, to their function in plant cells.
Animals and plant tissues undergo many different forms of development during their respective life cycles. Therefore, protein synthesis in each will be regulated according to that species. In addition, plant cells are totipotent, a single cell can regenerate into a whole plant. This is not the case with animal cells in culture. Therefore data derived from animal tissue culture systems cannot be extrapolated to include the functions of antisense messages in plant protoplast systems.
It is also not clear at what stage in protein synthesis that antisense action occurs. The antisense complementary strand may work on primary transcript (mRNA precursor) or either on the processing and/or the transport of the message from the nucleus into the cytoplasm. In transformed animal cells, interaction have been noted precisely in the nucleus where RNA:RNA hybrids formed between the mRNA and the complementary antisense RNA (3). These hybrids have never been found in plant systems.
Applicant has developed an expression system for antisense RNAs having particular use in plant biology. It utilizes a constitutive promoter element which is expressed in all plant tissues and should therefore be capable of inactivating a wide variety of gene functions.